Human coronaviruses (HCV) in two serogroups represented by HCV-229E and HCV-OC43 are an important cause of upper respiratory tract infections. Here we report that human aminopeptidase N, a cell-surface metalloprotease on intestinal, lung and kidney epithelial cells, is a receptor for human coronavirus strain HCV-229E, but not for HCV-OC43. A monoclonal antibody, RBS, blocked HCV-229E virus infection of human lung fibroblasts, immunoprecipitated aminopeptidase N and inhibited its enzymatic activity. HCV-229E-resistant murine fibroblasts became susceptible after transfection with complementary DNA encoding human aminopeptidase N. By contrast, infection of human cells with HCV-OC43 was not inhibited by antibody RBS and expression of aminopeptidase N did not enhance HCV-OC43 replication in mouse cells. A mutant aminopeptidase lacking the catalytic site of the enzyme did not bind HCV-229E or RBS and did not render murine cells susceptible to HCV-229E infection, suggesting that the virus-binding site may lie at or near the active site of the human aminopeptidase molecule.

To determine the primary structure of CD13, a 150-kD cell surface glycoprotein originally identified on subsets of normal and malignant human myeloid cells, we isolated the complete sequences encoding the polypeptide in overlapping complementary DNA (cDNA) clones. The authenticity of our cDNA clones was demonstrated by the ability of the coding sequences, subcloned in a retroviral expression vector, to mediate expression of bona fide CD13 molecules at the surface of transfected mouse fibroblasts. The nucleotide sequence predicts a 967 amino acid integral membrane protein with a single, 24 amino acid hydrophobic segment near the amino terminus. Amino-terminal protein sequence analysis of CD13 molecules indicated that the hydrophobic segment is not cleaved, but rather serves as both a signal for membrane insertion and as a stable membrane-spanning segment. The remainder of the molecule consists of a large extracellular carboxyterminal domain, which contains a pentapeptide consensus sequence characteristic of members of the zinc-binding metalloprotease superfamily. Sequence comparisons with known enzymes of this class revealed that CD13 is identical to aminopeptidase N, a membrane-bound glycoprotein thought to be involved in the metabolism of regulatory peptides by diverse cell types, including small intestinal and renal tubular epithelial cells, macrophages, granulocytes, and synaptic membranes prepared from cells of the central nervous system.

Quiescent mouse NIH3T3 cells expressing a transduced human c‐fms gene encoding the receptor for colony stimulating factor‐1 (CSF‐1) were stimulated with mitogenic concentrations of platelet‐derived growth factor (PDGF) or CSF‐1. Immunoprecipitated phospholipase C‐gamma (PLC‐gamma) was phosphorylated on tyrosine and calcium was mobilized following treatment of intact cells with PDGF. In contrast, only trace amounts of phosphotyrosine were incorporated into PLC‐gamma and no intracellular calcium signal was detected after CSF‐1 stimulation. Similarly, CSF‐1 treatment did not stimulate phosphorylation of PLC‐gamma on tyrosine in a CSF‐1‐dependent. SV40‐immortalized mouse macrophage cell line that expresses high levels of the CSF‐1 receptor. In fibroblasts, antiserum to PLC‐gamma co‐precipitated a fraction of the tyrosine phosphorylated form of the PDGF receptor (PDGF‐R) after ligand stimulation, implying that phosphorylated PDGF‐R and PLC‐gamma were associated in a stable complex. Pre‐treatment of cells with orthovanadate also led to tyrosine phosphorylation of PLC‐gamma which was significantly enhanced by PDGF, but not by CSF‐1. Thus, although the PDGF and CSF‐1 receptors are structurally related and appear to be derived from a single ancestor gene, only PDGF‐induced mitogenesis in fibroblasts correlated with tyrosine phosphorylation of PLC‐gamma.

fms genes encoding either wild-type or constitutively activated colony-stimulating factor 1 receptors (CSF-1R) were introduced by retroviral infection into long-term mouse lymphoid cultures. Four early pre-B-cell lines transformed by the feline v-fms oncogene underwent spontaneous and irreversible differentiation to macrophages when transferred from RPMI 1640 to Iscove modified Dulbecco medium. Expression of wild-type human CSF-1R in early pre-B cells conferred no proliferative advantage unless human CSF-1 was added to the culture medium. A clonal, factor-dependent early pre-B-cell line (D1F9), selected for continuous growth on NIH 3T3 cell feeder layers producing human CSF-1, could be maintained in RPMI 1640 medium containing interleukin-7 (IL-7) but also differentiated to macrophages when grown in Iscove modified Dulbecco medium containing human CSF-1. The macrophages retained parental immunoglobulin gene rearrangements and proviral insertions, lost B-cell antigens, expressed butyrate esterase and MAC-1, were actively phagocytic, and no longer survived in IL-7. Unlike factor-independent v-fms transformants, the irreversible commitment of D1F9 cells to differentiate in the macrophage lineage could be suppressed by IL-7, depended on human (but not mouse) CSF-1, and was inhibited by an antibody to human CSF-1R. Signals mediated by transduced CSF-1R can therefore play a deterministic role in cell differentiation.

We previously found that the myeloid cell surface glycoprotein CD13 (gp150) is identical to aminopeptidase N (EC 3.4.11.2), a widely distributed membrane-bound, zinc-dependent metalloprotease with an extracellular enzymatic domain that cleaves N-terminal amino acid residues from oligopeptides (J Clin Invest 83:1299, 1989). As a first step toward defining the function of this molecule on myeloid cells, we assessed cell surface-associated N-terminal peptidase activity by sensitive spectrophotometric measurements of the cleavage of p- nitroanilide amino acid derivatives. Aminopeptidase activity detected on the surface of normal and malignant hematopoietic cells coincided with the level of cell surface CD13 expression as measured by flow cytometry. The enzyme was specifically inhibited by the zinc-binding metalloprotease inhibitors, bestatin, 1,10-phenanthroline, or 2.2′- dipyridyl, but was not affected by several inhibitors of other classes of proteases. Aminopeptidase activity was demonstrated for CD13 molecules specifically immunoprecipitated from the surface of CD13- positive cells and was blocked by the metalloprotease inhibitor 1,10- phenanthroline. Moreover, cell surface aminopeptidase activity was partially inhibited when viable cells were incubated with two of a panel of 11 monoclonal antibodies (MoAbs) known to be specific for extracellular epitopes of human CD13. This inhibition was apparent in the absence of detectable downmodulation of CD13 molecules from the cell surface, suggesting that these MoAbs either physically interfere with substrate binding or alter the zinc-coordinating properties of aminopeptidase N molecules. Aminopeptidase N could play an important role in modulating signals generated by peptides at the surface of myeloid cells, either by removing key N-terminal residues from active peptides or by converting inactive peptides to active forms. The inhibitory antibodies used in this study should prove useful in delineating the physiologic roles of CD13/aminopeptidase N on normal and malignant myeloid cells.

DNA from the human myeloid cell line HL-60 was cotransfected with the cloned thymidine kinase (tk) gene of herpes simplex virus into tk-deficient mouse L cells. tk-positive recipients expressing antigens detected on HL-60 cells were isolated with a fluorescence-activated cell sorter by use of a panel of monoclonal antibodies that detect epitopes on both normal and malignant myeloid cells. Independently sorted populations of transformed mouse cells showed concordant reactivities with four of the monoclonal antibodies in the panel (DU-HL604, MY7, MCS.2, and SJ-D1), which suggested that these antibodies reacted to products of a single human gene. A second round of DNA transfection and cell sorting was performed with donor DNA from primary transformants. Two different dominant selection systems were used to isolate secondary mouse L cell and NIH/3T3 cell transformants that coexpressed the same epitopes. Analysis of cellular DNA from secondary mouse cell subclones with a probe specific for human repetitive DNA sequences revealed a minimal human DNA complement containing a characteristic set of restriction fragments common to independently derived subclones. Two glycoproteins, of 130,000 (gp130) and 150,000 (gpl50) mol wt, were specifically immunoprecipitated from metabolically labeled lysates of mouse cell transformants and were shown to contain V35SImethionine-labeled tryptic peptides identical to those of analogous glycoproteins expressed in the donor human myeloid cell line.Kinetic and biochemical analyses established that gpl3O is a precursor that differs in its carbohydrate moiety from gpl50, the mature form of the glycoprotein detected on the cell surface. The isolation of human gene sequences encoding gpl50 in a mouse cell genetic background provides the possibility of molecularly cloning the gene and represents a general strategy for isolating human genes encoding differentiation-specific cell surface antigens.

DNA from a tertiary mouse cell transformant containing amplified human sequences encoding a human myeloid membrane glycoprotein, gpl50, was used to construct a bacteriophage lambda library. A single recombinant phage containing 12 kilobases (kb) ofhuman DNA was isolated, and molecular subclones were then used to isolate the complete gplS0 gene from a human placental genomic DNA library. The intact gpl50 gene, assembled from three recombinant phages, proved to be biologically active when transfected into NIH 3T3 cells. Molecular probes from the gpl5O locus annealed with a 4.0-kb polyadenylated RNA transcript derived from human myeloid cell lines and from tertiary mouse cell transformants. The gpl50 gene was assigned to human chromosome 15, and was subchromosomally localized to bands q25-26 by in situ hybridization. The chromosomal location of the gpl50 gene coincides cytogenetically with the region assigned to the c-fes proto-oncogene, another human gene specifically expressed by myeloid cells.

The myeloid cell-surface glycoprotein CD13/aminopeptidase N (APN; EC 3.4.11.2) contains a pentapeptide (HExxH) in its extracellular domain that is characteristic of many zinc-dependent metalloproteinases. This region contains residues important for zinc binding and constitutes part of the catalytic domain of several metalloproteases. We deleted an internal fragment of 117 base pairs (bp) from the human CD13/APN cDNA, resulting in an in-frame deletion that included the sequences coding for this pentapeptide motif. The mutant cDNA was subcloned into a retroviral expression vector, and polypeptides encoded by the altered cDNA were expressed in transfected murine NIH-3T3 fibroblasts. The mutant CD13/APN molecules lacked enzymatic activity, and their intracellular processing to the cell surface was retarded by comparison with normal CD13/APN polypeptides. The mutant molecules also lacked epitopes required for binding of four of 19 CD13-specific monoclonal antibodies (MoAbs) tested in flow cytometric assays. Each of the four MoAbs also inhibited the enzymatic activity of wild-type APN molecules, suggesting that these antibodies may inhibit aminopeptidase activity by interfering with the enzyme's zinc-coordinating properties. Cells engineered to express mutant CD13/APN polypeptides at the cell surface provide a tool for defining the physiologic role of this enzyme on normal and malignant myeloid cells and marrow stromal cells.